The effects of the FASN blocker C75 on cell growth and survival (A, E, and F), on expression/phosphorylation of PI3K and MAPK signaling proteins and on protein ubiquitination (B–D) are not restricted to A2780 ovarian carcinoma cells. The effects of the FASN blocker C75 on cell growth and survival (A, E, and F), on expression/phosphorylation of PI3K and MAPK signaling proteins and on protein ubiquitination (B–D) are not restricted to A2780 ovarian carcinoma cells. A, three additional ovarian carcinoma cell lines (OVCAR-3, SKOV-3, and HEY) were exposed for 72 hours to 0 to 20 μg/mL C75 and were subjected to cell growth analysis using a formazan dye assay as described in Materials and Methods. Mean ± SD, n = 3, Student t test: 1, OVCAR-3, P < 0.001; 2, SKOV-3 P < 0.001; 3, HEY P < 0.001 versus 0 μg/mL C75 (control). B, the ovarian carcinoma cells were exposed for 72 hours to vehicle (≤0.1% DMSO) or to C75 (OVCAR-3, 6 μg/mL; SKOV-3, 20 μg/mL; HEY, 15 μg/mL) and were subjected to SDS-PAGE followed by Western blot analysis using antibodies against the total or the phosphorylated (p-) forms of AKT, S6, or ERK1/2, or against ubiquitin. β-Actin was used as loading control. One representative experiment. C, bands from autoradiographs were quantified by scanning densitometry. Each protein and p-protein band and the ubiquitinated proteins were normalized to β-actin. In addition, each p-protein band was also normalized to its corresponding total protein band. The resulting ratios (protein/actin, p-protein/actin, p-protein/protein) represent measures for expression, phosphorylation, and specific phosphorylation/activity of that particular protein, and for nonspecific protein ubiquitination, respectively. All calculated ratios were related to the ratios obtained from vehicle-treated control cells, which have been arbitrarily set at 1.0 (horizontal dashed line). Note that the y-axis has a log scale. Mean ± SD, n = 3, Student t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control. D, AKT is a direct client protein of C75-induced ubiquitination. Lysates of untreated (0.1% DMSO, 48 hours) and C75-treated (20 μg/mL, 48 hours) SKOV-3 cells were immunoprecipitated (IP) with antibodies against AKT or β-actin, respectively, subjected to SDS-PAGE and immunoblotted with antibodies against ubiquitin, AKT, or β-actin. β-Actin was used as loading control. Note: a C75-dependent increase of ubiquitinated AKT can be seen despite concurrent downregulation of the AKT protein steady-state level. E and F, C75 induces cell death in ovarian carcinoma cells. Treatment of SKOV-3 cells for 72 hours with C75 lowers the viable and elevates the nonviable cell population in a dose-dependent manner as shown by trypan blue dye exclusion (E). Western blot analyses for expression of caspase-3 and cleaved PARP-1 shows that OVCAR-3, SKOV-3, and HEY cells undergo apoptosis when exposed for 48 hours to 6, 20, or 15 μg/mL C75, respectively. β-Actin was used as loading control (F). Katharina Tomek et al. Mol Cancer Res 2011;9:1767-1779 ©2011 by American Association for Cancer Research